Mutation Research, 208 (1988) 17-19 17 Elsevier

MTRL 098

A sensitive assay for 6-thioguanine-resistant lymphocytes

Amos Norman, J. Cameron Mitchell and Keisuke S. Iwamoto

Departments of Radiological Sciences and Radiation Oneology and Jonsson Comprehensive Cancer Center, Universily o! Cal~/brnia, Los Angeles, CA 90024 (U.S.A.)

(Accepted 23 December 1987)

Keyword;: 6-Thioguanine-resistant lymphocytes, assay for; Lymphocytes, 6-thioguanine-resistant, assay for; Cytochalasin B.

Summary

We have defined variants (V) resistant to 6-thioguanine (TG) by their ability to divide at least once during

a 72-h incubation in a medium containing 0.2 mM TG. We blocked cytokinesis by adding cytochalasin B (CB) after 30 h in culture. The cells that had undergone nuclear division were identified by their content of 2 or more nuclei. The long incubation period allowed slow growing V to be counted. As a result we scored an order of magnitude more V than have been reported in assays using the conventional 30-40 h culture

times. In the "r-ray dose range of 0-0.5 Gy we scored 80 V per 1000 surviving lymphocytes per Gy - - a result some two orders of magnitude larger than has been reported previously.

The assay of variants (V) introduced by Strauss

and Albertini (1979) uses the incorporation of tritiated thymidine into the DNA of human peripheral lymphocytes during about a 30-h in- cubation in the precence of 6-thioguanine (TG) to

identify the V. The short incubation time was chosen to preclude cell division during culture.

One consequence, however, was to discriminate against the slow growing V. It seemed likely to us that the non-linear dose-response curve reported by Evans and Vijayalaxmi (1981) for X-ray- induced V might well be due, in part, to such discrimination. Moreover this factor would lead to

Correspondence: Dr. Amos Norman, Warren Hall, UCLA, 900 Veteran Ave., Los Angeles, CA 90024 (U.S.A.) (213) 825-7811.

a possibly large underestimate of the total V in-

duced by radiation or other mutagens. For that reason we developed an assay for V that identifies V by their nuclear division after a 72-h incubation.

Materials and methods

Blood was obtained by venipuncture from 3 nor- mal adults. The heparinized blood was centrifuged and the plasma and buffy coat layers were saved. Cultures were set up by resuspending the buffy coat cells in a medium consisting of RPMI 1640, 15°70 autologous plasma, and 1°70 P H A made up to the original blood volume. After 30 h of incuba- tion cytochalasin B (CB) was added to two sets of cultures, one containing 0.2 mM TG. The cultures were incubated at 38°C for 72 h and then the cells

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were fixed and stained with May-Grunwald/ Giemsa.

The cell suspensions were exposed to graded doses of cobalt-60 3,-radiation prior to incubation.

Variants per 1000 survivors were defined as the number of multinucleate cells per 1000 lym- phocytes in culture with TG divided by the number of multinucleate cells per 1000 lymphocytes in culture without TG.

Results

Fig. 1 presents the results of representative ex- periments. The main graph shows the increase of V with dose of 3,-rays for one donor. The error bars are calculated f rom the assumption of Poisson statistics for counting multinucleate cells. The inset shows the increase in multinucleate cells per 1000 total lymphocytes for 3 donors. The solid circles represent the same donor as in the main graph; the larger error bars in the main graph are the result of dividing 2 sets of counts. As can be seen the fraction of multinucleate cells up to 3, doses of 0.5 Gy increases among the total lym- phocytes. This increase continues but at a slower rate when the variants per surviving lymphocytes are calculated.

The 3 donors displayed V at zero 3, dose that in- creased with their age (23 for triangles, 30 for

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if:

280

240

2OO

16C

120

8O

4O

I.tO 2'.0 3'.0 DOSE (Gy)

Fig. I. 7-ray dose dependence of V (see text).

i 4 . 0

circles and 64 for squares). This is consistent with reports of the V dependence on donor age (Evans

and Vijayalaxmi, 1981). There is also an indication that the sensitivity to radiation, measured by the slope of the dose-response curve increases with age; but that needs confirmation.

The yield of V at zero dose, approximately 20 per thousand surviving lymphocytes, is one to two orders of magnitude larger than reported by Strauss and Albertini (1979) and Evans and Vi- jayalaxmi (1981). The radiation-induced increase in V over the first 0.5 Gy is 80 per 1000 surviving lymphocytes per Gy, some two orders of magnitude larger than the reports of Evans and Vi-

jayalaxmi (1981) for radiation-induced resistance to 8-azaguanine in human lymphocytes.

We scored micronuclei (MN) in the binucleate cells and found (data not shown) a monotonic in- crease with increasing TG concentration and with increasing radiation dose. However, the yield of MN at 1 Gy and 0.2 mM TG was only about 2 per 100 cells, suggesting that the chromosome aberra- tions that lead to MN are not a large factor in radiation-induced V.

Discussion

Our data are consistent with the findings of Amneus and Eriksson (1986) who showed an order of magnitude more V at the beginning than at the end of the S phase in short-term culture. However, our interpretation unlike theirs is that this is due to the slow growth of many V which are detected by allowing them longer growth times. Without blocking cell division, however, it is not feasible to take account of the increase in V due to cell divi- sion. One possible way to block division is with colcemid; however, metaphase cells do not survive well long periods in colcemid (Sasaki and Norman, 1966). We chose, therefore, to block cytokinesis with CB instead, because of evidence that cells do survive well in CB. It should be noted, however, that CB does slow the progression of cells through the cycle (Mitchell and Norman, 1987); and we did not get satisfactory growth when CB was added much before 30 h in culture.

W e note , f ina l ly , t h a t th is m e t h o d fo r a s say ing

V lends i t se l f wel l to s co r ing by f l o w c y t o m e t r y . It

is essent ia l , h o w e v e r , to p r o d u c e s ingle cell suspen-

s ions wh ich has p r o v e d d i f f i cu l t w i th o u r m e t h o d

fo r c u l t u r i n g cells.

References

Amneus, H., and L. Eriksson (1986) The frequency of 6-thioguanine resistant human peripheral blood lymphocytes as determined by flow cytometry and clonal propagation, Muta;ion Res., 173, 61-66.

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Evans, H.J., and Vijayalaxmi (1981) Induction of 8-azaguanine resistance and sister-chromatid exchange in human lym- phocytes exposed to mitomycin c and X-rays in vitro, Nature (London), 292, 601-605.

Mitchell, J.C., and A. Norman (1987) The induction of micronuclei in human lymphocytes by low doses of radia- tion, Int. J. Radiat. Biol., in press.

Sasaki, M.S., and A. Norman (1966) Proliferation of human lymphocytes in culture, Nature (London), 210, 912-914.

Strauss, G.H., and R.J. Albertini (1979) Enumeration of 6-thioguanine resistant peripheral blood lymphocytes in man as a potential test for somatic cell mutation arising in vivo, Mutation Res., 61, 353-379.

Communicated by R.J. Preston